1. Field of the Invention
The present invention relates to novel polypeptides useful for treating hypomineralization defects of bone and teeth, such as that leading to rickets, osteomalacia, age-related bone-mineral loss disorders, and osteoporosis.
2. Description of Related Art
PHEX (an acronym for phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) is predominantly expressed in osteoblasts, osteocytes, and odontoblasts. PHEX is a zinc metalloendopeptidase integral membrane glycoprotein having a short cytoplasmic N-terminal region, a single small hydrophobic transmembrane domain, and a large extracellular C-terminal region with a zinc-binding motif having the sequence HEFTH (SEQ. ID NO. 1). Defects in the PHEX gene are primarily responsible for X-linked hypophosphatemic rickets (“HYP”). The disease is characterized by low serum phosphorus, dis-regulated or inappropriately normal serum 1,25 vitamin D3, elevated serum alkaline phosphatase and severe hypomineralization defects of bone and teeth leading to rickets and osteomalacia.
PHEX contains key residues for catalytic activity of small peptides, but the substrate for its oligopeptidase activity remains to be identified. As discussed more fully below, PHEX also functions as protein ligand, most notably for Matrix extracellular phosphoglycoprotein (“MEPE”) and a protease-resistant MEPE-derived peptide known as the ASARM peptide.
MEPE belongs to a distinct family of bone-dentin salivary proteins that have recently been named short integrin binding ligand interacting glycoproteins (“SIBLINGS”). Evidence suggests that MEPE inhibits phosphate uptake and mineralization in-vivo and in-vitro and is involved in extracellular matrix mineralization (minhibin). See Dobbie et al., Infusion of the bone-derived protein MEPE causes phosphaturia in rats (Abstract), J. Am. Soc. Nephrol. 14:468A (2003); Rowe et al., MEPE has the properties of an osteoblastic Phosphatonin and Minhibin, Bone 34:303-319 (2004). For example, MEPE-mediated in vivo phosphaturia in rodents can be induced via bolus administration or infusion. Further, MEPE null-mutant mice have increased bone mass, resistance to aging associated trabecular bone loss, increased mineralization-opposition rate (“MAR”), and a dramatically accelerated mineralization rate in ex vivo osteoblast cultures. See Gowen et al., Targeted Disruption of the Osteoblast/Osteocyte Factor 45 Gene (OF45) Results in Increased Bone Formation and Bone Mass, J. Biol. Chem. 278:1998-2007 (2003).
Recent work has shown that MEPE binds to PHEX via the PHEX zinc-binding motif and a carboxy-terminal MEPE motif known as the acidic-serine-aspartate-rich MEPE associated motif (“ASARM”). See Rowe et al., Surface Plasmon Resonance (SPR) confirms MEPE binds to PHEX via the MEPE-ASARM-motif: A model for impaired mineralization in X-linked rickets (HYP), Bone 36:33-46 (2005). Cleavage by the protease cathespin-B, which is expressed in osteoblasts, results in the release of the ASARM peptide. When liberated as a phosphorylated peptide (2.2 kDa) in its free form, this small, acidic, highly charged ASARM peptide is a potent inhibitor of mineralization and phosphate uptake. See Bresler et al., Serum MEPE-ASARM peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets, J. Endocrinol. 183:R1-9 (2004); Dobbie et al., Infusion of the bone-derived protein MEPE causes phosphaturia in rats (Abstract), J. Am. Soc. Nephrol. 14:468A (2003). As evidence, researchers have shown excess circulating levels of either COOH-terminal MEPE or ASARM-peptides present in HYP and oncogenic hypophosphatemic osteomalacia (“OHO”) patients. See Bresler et al., Serum MEPE-ASARM peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets, J. Endocrinol. 183:R1-9 (2004); de Beur et al., Matrix Extracelluler Phosphoglycoprotein (MEPE), fragments circulate in excess in patients with Tumor-induced Osteomalacia (TIO) and X-linked Hypophosphatemic Rickets (XLH), J. Bone Miner. Res. 19:F479 (Abstract) S101 (2004). It has also been confirmed that HYP-mice kidneys have excess levels of ASARM-peptide epitopes in regions consistent with the proximal convoluted tubules. See Bresler et al., Serum MEPE-ASARM peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets, J. Endocrinol. 183:R1-9 (2004).
The ASARM cleavage site of MEPE is highly conserved in all cloned species (mouse, rat, monkey, and human), and the ASARM peptide is also extraordinarily resistant to a vast array of proteases, including carboxypeptidases (B/T), cathespins-(BDGK), trypsin, granzyme A, papain, pepsin, kallikrein, plasmin, nardilysin, NEP, and ECEL1/DINE.
Researchers have recently theorized that PHEX protects MEPE from cathepsin proteolysis. See Rowe, The wrickkened-pathways of FGF23, MEPE and PHEX, Crit. Rev. Oral Biol. Med 15:264-281 (2004). It has been theorized that the MEPE-PHEX interaction in vivo may therefore prevent proteolytic release of the protease-resistant ASARM peptide by protecting MEPE from cathepsin-B and general protease degradation. See Rowe, The wrickkened-pathways of FGF23, MEPE and PHEX, Crit. Rev. Oral Biol. Med 15:264-281 (2004). A summary of the proposed PHEX-MEPE-ASARM-cathepsin pathway is illustrated in FIG. 1.
Both the PHEX gene and MEPE have been cloned, and their sequence disclosed. See GenBank CAA712528; Rowe et al., Distribution of mutations in the PEX gene in families with X-linked hypophosphatemic rickets (HYP), Human Mole. Gen., 6, 539-549 (1997); Francis et al., Genomic organization of the human PEX gene mutated in X-linked dominant hypophosphatemic rickets, Gen. Res. 7(6):573-585 (1997); Rowe et al., MEPE, a new gene expressed in bone-marrow and tumours causing osteomalacia, Genomics. 67 (1):54-68 (2000); Argiro et al., Mepe, the gene encoding a tumorsecreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone, Genomics 74:342-51 (2001); Petersen et al., Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD containing protein that is highly expressed in osteoblasts and osteocytes, J. Biol. Chem. 275.36172-80 (2000); Rowe, U.S. Pat. No. 6,673,900 entitled “Polypeptide hormone-phosphatonin”; Crine et al., U.S. Pat. No. 6,790,649 entitled “Composition Methods and Reagents for the Synthesis of a Soluble Form of Human PHEX.” MEPE maps to 4q21 and shares many features with the SIBLINGS, including RGD motifs, protein glycosylation, phosphorylated residues, similar genomic structures, and the ASARM motif.
The present invention is directed to compositions and methods for promoting bone mineralization and phosphate uptake by targeting a specific event in the PHEX-MEPE-ASARM-protease pathway. In particular, novel ASARM binding compounds are provided. These ASARM binding compounds are polypeptides that mimic key binding residues of the PHEX protein capable of binding to the full-length MEPE or the ASARM fragment, thus reducing the concentration of biologically active ASARM peptides in vivo and in vitro and/or protecting MEPE from proteolysis and release of free ASARM-peptide.
Administration of these novel polypeptides results in improved bone mineralization and renal phosphate uptake.